Journal of the Marine Microscopy of crustacean cuticle: formation of Biological Association of the United Kingdom a flexible extracellular matrix in moulting sea slaters Ligia pallasii cambridge.org/mbi J. Štrus1,M.Tušek-Žnidarič2, U. Repnik3, A. Blejec2 and A. Summers4 1Department of Biology, University of Ljubljana, SI-1000 Ljubljana, Slovenia; 2National Institute of Biology, SI-1000 Original Article Ljubljana, Slovenia; 3Department of Biosciences, University of Oslo, NO-0316 Oslo, Norway and 4University of Washington, Friday Harbor Laboratories, Washington State, USA Cite this article: ŠtrusJ,Tušek-Žnidarič M, Repnik U, Blejec A, Summers A (2019). Abstract Microscopy of crustacean cuticle: formation of a flexible extracellular matrix in moulting sea Structural and functional properties of exoskeleton in moulting sea slaters Ligia pallasii from slaters Ligia pallasii. Journal of the Marine the Eastern Pacific coast were investigated with CT scanning and electron microscopy. Biological Association of the United Kingdom Ultrastructure of preecdysial and postecdysial cuticular layers was described in premoult, 99,857–865. https://doi.org/10.1017/ S0025315418001017 intramoult and postmoult animals. Cuticle is a flexible extracellular matrix connected to the epidermal cells through pore channels. During premoult epicuticle and exocuticle are Received: 26 April 2018 formed and during intramoult and postmoult endocuticular lamellae are deposited and the Revised: 5 September 2018 cuticle is progressively constructed by thickening and mineralization. Cuticle permeability, Accepted: 26 October 2018 flexibility and waterproofing capacity change accordingly. Elaboration of epicuticular scales con- First published online: 4 December 2018 nected to an extensive network of nanotubules, establish its anti-adhesive and hydrophobic Key words: properties. Labelling with gold conjugated WGA lectins on Tokuyashu thawed cryosections Cuticle ultrastructure; micro CT scanning; exposes differences in chitin content between exocuticle and endocuticle. Histochemical staining moult cycle; Oniscidea; SEM; TEM; terrestrial of cuticle shows presence of acidic carbohydrates/glycoconjugates and lipoproteins in epicuticu- isopods lar layer. Chitin microfibrils are formed at the microvillar border of epidermal cells with abun- Author for correspondence: dant Golgi apparatus and secretory vesicles. Numerous spherules associated with nanotubules J. Strus, Email: [email protected] were observed in the ecdysial space in intramoult animals. The mineral component of the cuticle as visualized with CT scanning indicates progressive mineral resorption from the posterior to the anterior half of the body in premoult animals, its translocation from the anterior to posterior part during intramoult and its progressive deposition in the posterior and anterior exoskeleton during postmoult. Cuticle of sea slaters is a unique biocomposite and biodynamic material constantly reconstructed during frequent moults, and adapted to specific physical and biotic conditions of the high intertidal rocky zone. Introduction Crustacean cuticle is an extracellular matrix secreted by epidermal and gut ectodermal cells. The structure and function of the cuticles has been studied extensively, and its structural and compositional features have been described in different body parts of adults and during development in different crustacean groups (Storch & Štrus, 1989; Strus & Storch, 1991; Wägele, 1992; Strus et al., 1995; Ziegler, 1997; Žnidaršič et al., 2010; Luquet, 2012; Dillaman et al., 2013;Mraket al., 2014; Roer et al., 2015). Cuticle is a complex mineralized chitinous-proteinaceous matrix with a multilayered structure. In crustaceans it is constantly being remodelled by exuviation during reproduction, development and growth. Several studies describe cuticle structure in terrestrial isopods during embryonic development and during growth in adults. They mostly focus on time-dependent events of cuticle secretion during development of embryos and larvae (Wolff, 2009; Milatovič et al., 2010; Žnidaršič et al., 2012;Mraket al., 2014, 2017) or on cuticle structure and biomineralization during moult cycle (Ziegler, 1997; Hild et al., 2008; Ziegler et al., 2017; Žnidaršič et al., 2018). There are © Marine Biological Association of the United also several reports about terrestrial isopod cuticle structure and function from the viewpoint Kingdom 2018. This is an Open Access article, distributed under the terms of the Creative of adaptation to lifestyles in different habitats, mostly with regard to epicuticle ornamentation, Commons Attribution licence (http:// number and thickness of cuticular lamellae and intensity of mineralization (Schmalfuss, 1984; creativecommons.org/licenses/by/4.0/), which Hild et al., 2009; Seidl et al., 2011; Hornung, 2011; Luquet, 2012; Vittori & Štrus, 2014; Wood permits unrestricted re-use, distribution, and et al., 2017). reproduction in any medium, provided the original work is properly cited. Amphibious sea slaters Ligia pallasii Brandt, 1833 are very convenient for studies of cuticle structure and formation due to frequent biphasic moulting and poorly mineralized exocuticle in contrast to crab cuticle. Cuticle of sea slaters is an important surface barrier, which prevents ion and water loss and enables mobility and communication in the terrestrial environment. Its structural and functional adaptations show features related to transition from marine to land habitats (Carefoot & Taylor, 1995; Strus & Compere, 1996; Strus & Blejec, 2001). Cuticle of strictly terrestrial woodlice is well described and its structure reflects specific adaptations to different land lifestyles (Hornung, 2011; Seidl & Ziegler, 2012; Vittori et al., 2012, 2017). Imaging of cuticle is a demanding procedure, due to its compositional and structural instabil- ity during the moult cycle. Adequate preparation of tissues that secrete cuticle is of utmost importance for preservation of structural and chemical components. Microscopic structure of crustacean cuticles at different resolution levels is presented in various papers mostly Downloaded from https://www.cambridge.org/core. IP address: 170.106.35.234, on 25 Sep 2021 at 03:10:04, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0025315418001017 858 J. Štrus et al. Fig. 1. Light micrographs and micro CT of moulting sea slater Ligia pallasii.(A) Intramoult animal with shed posterior half of the exoskeleton. (B) Front part of the postmoult animal with shed anterior half of the exoskeleton. (C) Dorsal view of an intra- moult animal with shed posterior half of the exoskel- eton, showing progressive resorption of mineral from the anterior half of the old cuticle in posterior-anter- ior direction. (D) Lateral view of the intramoult ani- mal with mineral prevailing in the head with antennae and mouthparts. The separation line between anterior and posterior half of the body is marked by an arrow. Scale bars: 5 mm. focused on studies of presence and distribution of biominerals in Center at Friday Harbor Labs. The images were reconstructed the extensive chitinous-proteinaceous matrix (Dillaman et al., and visualized with Amira (Thermo Fisher Scientific). 2005; Ziegler et al., 2006; Luquet, 2012). Histological methods, fluorescence microscopy with acridine orange and electron Sample preparation for electron microscopy microscopy combined with various analytical techniques are the most frequent approaches in studies of cuticle structure and com- Animals were dissected and dorsal parts of the anterior exoskel- position (Marlowe et al., 1994). A new approach of mineralized eton (tergites 3, 4) were fixed in 2% glutaraldehyde and 2% par- cuticle visualization in moulting terrestrial isopod Porcellio scaber aformaldehyde in 0.1 M HEPES buffer (Fixative 1) for a structural with SR micro CT shows that different parts of exoskeletal ele- analysis using a Hitachi S-4800 Field Emission Scanning Electron ments are involved at varying extents in mineral recycling during Microscope. Samples were prepared as described before (Vittori & the moult cycle (Ziegler et al., 2017). Štrus, 2014). Alternatively, samples were fixed in 4% paraformal- In this paper we introduce X-ray microtomography (micro dehyde in 0.1 M HEPES buffer (fixative 2) for chitin and protein CT) and transmission electron microscopy of wheat germ agglu- localization with TEM using Tokuyasu technique for sample tinin (WGA) lectin gold labelled Tokuyasu thawed cryosections preparation. For this, tergites were embedded in 12% bovine gel- for investigation of chemical composition of different cuticular atine and infiltrated with 2.3 M sucrose before they were cut into layers. The aim of this contribution is visualization of surface epi- smaller pieces, which were additionally infiltrated with sucrose for thelia and cuticle which they secrete during the moult cycle, using 24 h. Samples were mounted on metal pins and snap frozen in a non-invasive method of micro CT and electron microscopy of liquid nitrogen for cryo-sectioning in a Leica EM UC7 ultramicro- cryosections. Structural and compositional dynamics of cuticular tome. Sections (80–100 nm) were thawed and transferred to layers are presented during three different phases of the moult carbon- and formvar-coated copper grids. For immunogold label- cycle in Ligia pallasii and discussed based on known data about ling Wheat Germ Agglutinin (WGA)-biotin (Vector Laboratories) cuticle